RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
Methods and constructs for RNA-guided targeting of heterologous functional domains such as transcriptional activators to specific genomic loci.
1. A fusion protein comprising catalytically inactive CRISPR associated 9 (dCas9) protein linked to a Heterochromatin Protein 1 (HP1).
2. The fusion protein of claim 1 , wherein the catalytically inactive Cas9 protein is from S. pyogenes.
3. The fusion protein of claim 2 , wherein the catalytically inactive Cas9 protein comprises mutations at D10, E762, H983, or D986; and at H840 or N863.
4. The fusion protein of claim 3 , wherein the mutations are:
(i) D10A or D10N, and
(ii) H840A, H840N, or H840Y.
5. The fusion protein of claim 1 , wherein the heterologous functional domain is linked to the N terminus or C terminus of the catalytically inactive Cas9 protein, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.
6. The fusion protein of claim 1 , further comprising one or both of a nuclear localization sequence and one or more epitope tags on the N-terminus, C-terminus, and/or in between the catalytically inactive CRISPR associated 9 (Cas9) protein and the heterologous functional domain, optionally with one or more intervening linkers.
7. The fusion protein of claim 6 , wherein the epitope tag is c-myc, 6His, or FLAG.
8. A nucleic acid encoding the fusion protein of claim 1 .
9. An expression vector comprising the nucleic acid of claim 8 .
10. A method of decreasing expression of a target gene in a cell, the method comprising expressing the fusion protein of claim 1 in the cell, and one or more guide RNAs directed to the target gene.